蜡样芽孢杆菌分型方法研究进展

蜡样芽孢杆菌是一种食源性条件致病菌,在环境和食品中分布比较广泛,所引起的食物中毒常分为腹泻型和呕吐型。研究蜡样芽孢杆菌的分型方法对该菌的预防、预警、溯源及分子流行病学的调查都具有重要意义。目前,蜡样芽孢杆菌的分型方法主要包括噬菌体分型、生化分型等传统分型方法以及多位点序列分型、重复序列聚合酶链式反应分型、随机扩增多态性DNA分型、脉冲场凝胶电泳分型等分子分型方法。以蜡样芽孢杆菌为对象,综述其分型方法的研究进展,以期为该菌的分型提供一定参考。     

Listeria species in seafood: isolation and characterization of Listeria spp. from seafood in San Luis, Argentina

One hundred seafood samples (fish, squid and mussel) were collected from the Argentine Atlantic coast and screened for Listeria spp. The isolates were characterized by biochemical tests, serotyping, phage typing and macrorestriction enzymes analysis of DNA (pulsed-field gel electrophoresis). The overall frecuency (n=100) ofListeria spp. was 12%. Of the 12 isolated strains, three strains isolated from different squid samples were identified as L. monocytogenes and nine strains from fish, mussels and squid as L. innocua. All three L. monocytogenes strains belonged to serovar 4b; eight strains of L. innocua were serovar 6a and one strain of L. innocua was serovar 6b. All three L. monocytogenes strains, but only one of the nine L. innocua strains, were phage-typeable. One restriction profile was detected with Apa I and Asc I for L. monocytogenes strains and three with the same enzymes for L. innocua strains. The combination of these patterns allowed definition of four distinct groups within the 12 strains. The results of this study showed that phenotypic methods remain appropriate but that pulsed-field gel electrophoresis is useful for epidemiological purposes.     

食源性致病菌溯源分型技术研究进展

食源性致病菌是影响食品质量与安全的主要因素。及时和准确的菌株分型数据能够快速检测暴发集群,为正在进行的感染控制和公共卫生应对活动提供信息,对于食源性疾病的预防与爆发具有重要意义。近年来各种细菌分型方法取得了巨大进展,本文对几种常见食源性致病菌溯源分型技术进行综述,包括血清分型技术、噬菌体分型技术、脉冲场凝胶电泳技术、变性梯度凝胶电泳技术、细菌基因组重复序列PCR技术（Rep-PCR）、低频限制性切割位点PCR技术（IRS-PCR）、扩增片段长度多态性技术（AFLP）、限制性片段长度多态性技术（RFLP）、多位点序列分型技术（MLST）、核心基因组多位点序列分型技术（cgMLST）、单核苷酸多态性分型技术以及基质辅助激光解析电离飞行时间质谱技术（MALDI-TOF-MS）等。针对不同技术的原理对其所适用的环境进行阐述,并对分型溯源能力进行比较分析,为流行病学中食源性致病菌监测方案提供参考依据。     

Emergence and characterization of foodborne methicillin-resistant Staphylococcus aureus in Korea

A total of 165 Staphylococcus aureus strains, isolated from different food samples between 2003 and 2006, were tested for antimicrobial susceptibility. The mecA-positive methicillin-resistant S. aureus (MRSA) strains were further characterized by testing for various virulence genes and by molecular typing with multilocus sequence typing and pulsed-field gel electrophoresis. Of the 165 S. aureus isolates, 150 strains (90.9%) were resistant to at least one antibiotic while no strain was resistant to vancomycin. Four strains were resistant to both oxacillin and cefoxitin and were mecA positive. The mecA-positive MRSA strains were isolated from raw meat and fish samples (two beef samples and two fish samples) and were resistant to β-lactam antibiotics. Based on multilocus sequence typing analysis, the isolates were assigned to sequence type 1 (ST1), ST72, and an undetermined ST (ST72 slv). All four MRSA isolates were shown to be enterotoxigenic. The ST1 MRSA isolate harbored the sea-seh gene combination and the ST72 and ST72 slv MRSA strains harbored the seg-sei and the sea-seg-sei gene combinations, respectively. However, none of the MRSA isolates had the genes for Panton-Valentine leukocidin, toxic shock syndrome toxin 1, and exfoliative toxins. The pulsed-field gel electrophoresis patterns of the ST72 isolates in our study were highly similar, even though they were isolated from food samples in different years and from different regions of Korea.     

Pulsed-field gel electrophoresis for the discrimination of Oenococcus oeni isolates from different wine-growing regions in Germany

Reliable techniques are needed for the identification individual Oenococcus oeni strains with desirable flavor characteristics and to monitor the survival and contribution of inoculated and indigenous bacteria. Therefore, we investigated the suitability of pulsed-field gel electrophoresis (PFGE) for the discrimination of 65 O. oeni isolates from six different wine-producing regions in Germany. Among the restriction enzymes tested, genomic DNA digestions with Sfi I were most effective by displaying 56 (86%) different banding profiles. Our results underline the high capacity of PFGE for strain identification and differentiation. Cluster analysis of the DNA restriction patterns revealed no distinct region specificity of O. oeni populations.     

Genetic procedures for identification of enterotoxigenic strains of Staphylococcus aureus from three food poisoning outbreaks

Three food poisoning restaurant outbreaks due to Staphylococcus aureus, occurring during June-October 2002 in the Principality of Asturias (PA), Spain, provided the basis for investigating some aspects of the molecular epidemiology of this organism. The methods applied to identify strains and lineages included multiplex-polymerase chain reaction (PCR) to detect nine enterotoxin (se) genes, and three DNA fingerprinting procedures: pulsed-field gel electrophoresis (PFGE) with SmaI, randomly amplified polymorphic DNA (RAPD) with two selected primers, and plasmid restriction analysis with HindIII. Thirty-two isolates were differentiated into three non-se and 12 se strains, which were outbreak-specific, except for one that was represented in two of the outbreaks. In outbreak 1, the 16 food isolates analyzed had sec, seg and sei genes and generated a distinctive DNA fingerprint, being assigned to a single strain. This strain could be categorized as endemic in the PA and associated to manually handled dairy products and nasal carriers. In outbreak 2, the four food isolates analyzed fell into three strains, each one displaying a different se-gene profile (sea, sec and seg-seh-sei) and a distinctive DNA fingerprint. In outbreak 3, the five food isolates tested fell into four seg-sei strains generating identical RAPD but different PFGE and plasmid profiles, and one sea strain also collected from two nasal carriers. This last strain had also been found in manually handled vegetables in outbreak 2, and it belongs to a not very frequently found sea lineage in the PA. Multiplex-PCR to detect se genes together with the three applied DNA fingerprint typing procedures proved therefore to be useful tools in subclassifying S. aureus for epidemiological purposes.     

广西香港海鸥菌分离菌株脉冲场凝胶电泳分子分型研究

目的 探讨香港海鸥菌分子分型方法,了解广西水产品监测所分离的香港海鸥菌的相关性.方法 以NotⅠ限制性内切酶对2005年分离的香港海鸥菌酶切后进行脉冲电泳,用BioNumerics 5.1聚类分析获得电泳图谱.结果 7株香港海鸥菌分为6个分子型,其中从南宁分离的与从河池分离的2株香港海鸥菌高度同源,相似度达100％.结论 PFGE可应用于香港海鸥菌分子分型,有助于发现香港海鸥菌流行规律和传播途径,水鸟可能是香港海鸥菌传播环节的一种重要媒介.     

Phenotypic and genotypic characterization of salmonella Enteritidis isolated from two consecutive Food-Poisoning outbreaks in Sichuan,China

Salmonella enterica serotype Enteritidis (SE) is a primary pathogen that causes foodborne diseases in humans. Although whole-genome sequencing (WGS) -based typing analyses have been increasingly used to investigate food-poisoning outbreaks, they are rarely applied to the epidemiology of multiple Salmonella outbreaks in Sichuan, China. This study therefore isolated SE from patients and food of two consecutive food-poisoning outbreaks during 2020 in Sichuan, China. We tracked outbreak origin using epidemiological investigation, serotyping, antimicrobial susceptibility testing (AST), pulsed-field gel electrophoresis (PFGE), and WGS. We also determined phylogenetic relationships using PFGE, whole and core genome multilocus sequence typing (wg/cgMLST), and whole-genome single nucleotide polymorphism (wgSNP) analyses. Epidemiological investigations identified a correlation between cake consumption and food poisoning. Thirteen strains isolated from patients and one strain isolated from the cake were confirmed as SE. Among the 14 strains, only six shared the same AST pattern (AMP-AMS-Sul-STR). Isolates from patients and cakes were indistinguishable in PFGE results. All four methods, namely PFGE, wgMLST, cgMLST, and wgSNP were appropriate for bacterial typing in SE-related outbreak investigation. However, wgSNP can assign 12 SE strains from the first outbreak to one cluster and assign two SE strains from the second outbreak to another cluster, while PFGE, wgMLST, cgMLST did not successfully distinguish the SE strains from different outbreaks. Thus, we conclude that SNP-based phylogenetic analysis might be a viable method for differentiating SE strains at the outbreak level.     

Application of Random Amplified Polymorphic DNA (RAPD) and Pulsed-Field Gel Electrophoresis (PFGE) Analysis to Listeria monocytogenes: A Review of Methodology and Results

Random amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) analyses have been found to be powerful molecular methods for differentiating isolates of a given bacterial species. When applied to Listeria monocytogenes, both methods have been found highly effective in tracking isolates involved in food borne outbreaks of listeriosis and in identifying routes of contamination in food processing plants. Among the two methods, PFGE is considered somewhat superior in discriminatory power. However, the use of two or more independent random primers with RAPD is considered to result in a level of discrimination equal to that of PFGE. When results from both methods are combined, a maximum level of discrimination that exceeds that obtained with both methods independently can be achieved. Individually, both methods far exceed the discriminatory power of serotyping and phage typing of L. monocytogenes strains in that serotypes 1/2a, 1/2b, and 4b, represent over 90% of all human isolates, and phage typing at times has allowed typing of no more than about 50% of isolates. In addition, both RAPD and PFGE on occasion have been found to be superior to ribotyping, multilocus enzyme electrophoresis, and restriction enzyme analysis of L. monocytogenes isolates.     

苏州市食源性金黄色葡萄球菌耐药分析及基因分型

目的 对苏州市2014~2017年分离的食源性金黄色葡萄球菌进行耐药谱和分子分型分析, 初步建立PFGE(pulsed-field gel electrophoresis)分型数据库。方法 采用微量肉汤稀释法检测金黄色葡萄球菌(Staphylococcus aureus, SAU)对19种常用的抗生素的耐药性, 采用Sma I进行酶切, 脉冲场凝胶电泳进行分子分型。菌株指纹图谱用BioNumerics v6.6软件进行分析。结果 81株SAU中检出10株耐甲氧西林金黄色葡萄球菌(methicillin-resistance Staphylococcus aureus, MRSA), 检出率12.3%。有72株菌存在不同程度的耐药, 耐药率达到88.9%, 其中对青霉素的耐药率最高(82.7%), 其次是氨苄西林(66.7%), 其后是克林霉素和红霉素(均为50.6%)。耐受3种和3种以上药物的多重耐药菌株共38株, 多重耐药率达46.9%。MRSA的多重耐药率高于甲氧西林敏感型金黄色葡萄球菌(methicillin-sensitive Staphylococcus aureus, MSSA), 差异具有统计学意义(P<0.05)。除8株菌不可被PFGE分型外, 其余73株菌可以被分为37个不同型别, 其中有4个优势型别共27株菌, 在可分型菌株中占比达37.0%。这些同源菌株分离自不同时间和地点, 有些还存在一定程度的变异。结论 食源性金黄色葡萄球菌耐药现象普遍存在, 多重耐药率较高, PFGE型别较为多样但有优势型别存在, 耐药谱和PFGE型别关联性较差。     

Inhibition of Listeria monocytogenes by Carnobacterium spp. strains in a simulated cold smoked fish system stored at 4 degrees C.

Preservation of smoked salmon from bacterial spoilage, and especially from Listeria monocytogenes by bacteriocin producers is a promising challenge. Over a hundred lactic acid bacteria, isolated from commercial vacuum packaged cold smoked salmon, were screened for their antagonistic activity against L. innocua. Twenty-two strains were able to produce bacteriocin-like proteinaceous substances. These strains were characterized physiologically and biochemically as Carnobacterium strains. Three different groups were determined by pulsed-field gel electrophoresis after Sma I and Apa I DNA digestion. Peptidoglycan hydrolases patterns completed the characterization of these strains. All were confirmed as being Carnobacterium piscicola. Growth and bacteriocin production of three strains of each group and two well known bacteriocin producers (C. divergens V41 and C. piscicola V1) were tested in a simulated cold smoked fish system at 4 degrees C. These strains were able to reach 10(8) cfu ml(-1) in 21 days and to produce as much bacteriocin activities in the cold smoked fish system as in the rich media. Carnobacterium divergens V41 and C. piscicola V1 were the most effective strains in co-culture experiments, inhibiting L. monocytogenes as early as day 4, whereas C. piscicola SF668 inhibiting effect was observed at day 13. The potential for using such biopreservation treatments on whole smoked salmon is discussed.     

食源性病原菌分型方法研究进展

目前食源性病原菌常用的分型方法主要包括血清学分型、脉冲场凝胶电泳分型、多位点序列分型、多位点可变数目串联重复序列分型等, 在食源性病原菌的监测、传染源的追踪、流行病监测等方面具有重要意义。本文就食源性病原菌常用的分型方法及其应用情况进行综述。     

Molecular discrimination of new isolates of Bifidobacterium animalis subsp. lactis from reference strains and commercial probiotic strains

Fifteen new isolates from pig faeces were identified as Bifidobacterium animalis subsp. lactis by polymerase chain reaction (PCR) using primers specific for this subspecies. Ten of the isolates could be differentiated at strain level by randomly amplified polymorphic DNA (RAPD) analysis and pulsed-field gel electrophoresis (PFGE). These new strains were different from the type strain and other reference strains of this subspecies, and from all commercial dairy strains with probiotic functionality. Thus, possibly some of the isolates are potential candidates for new probiotic Bifidobacterium strains that can be unambiguously identified by RAPD-PCR and PFGE, which could be of interest for the food industry. In contrast, reference strains and commercial dairy strains revealed great genetic homogeneity, showing almost identical DNA fingerprints using RAPD-PCR and PFGE. Some reference strains and all commercial probiotic strains that had been originally designated as B. animalis or B. lactis have to be assigned to B. animalis subsp. lactis to be correctly labelled.     

脉冲场电泳在两起同期异地肠炎沙门菌食物中毒分子流行病学调查中的应用研究

目的 探讨两起相距210多公里乡镇同期发生的肠炎沙门菌食物中毒分离株之间的分子流行病学关系.方法 将两起食物中毒事件患者和剩余食物分离到的8株肠炎沙门菌进一步鉴定培养,挑取单个菌落增菌,用限制性内切酶Xba I消化酶切后进行脉冲场凝胶电泳,获得的分子分型指纹图谱,运用Bionumerics 5.1进行聚类分析.结果 指纹图谱聚类显示8株肠炎沙门菌分为两个分子型,D乡食物中毒分离到的5株肠炎沙门菌有相同的指纹图谱;P镇的3株分离株图谱也一致;两起食物中毒分离株图谱之间有4个条带的差异.结论两起食物中毒的暴发虽然都是肠炎沙门菌引起的,但具有不同的分子型别,食物中毒不是由同一种污染的食物源引起;脉冲场凝胶电泳可有效应用于食物中毒溯源分析及肠炎沙门菌分子流行病学研究.     

Strain typing of acetic acid bacteria responsible for vinegar production by the submerged elaboration method

Strain typing of 103 acetic acid bacteria isolates from vinegars elaborated by the submerged method from ciders, wines and spirit ethanol, was carried on in this study. Two different molecular methods were utilised: pulsed field gel electrophoresis (PFGE) of total DNA digests with a number of restriction enzymes, and enterobacterial repetitive intergenic consensus (ERIC) – PCR analysis. The comparative study of both methods showed that restriction fragment PFGE of SpeI digests of total DNA was a suitable method for strain typing and for determining which strains were present in vinegar fermentations. Results showed that strains of the species Gluconacetobacter europaeus were the most frequent leader strains of fermentations by the submerged method in the studied vinegars, and among them strain R1 was the predominant one. Results showed as well that mixed populations (at least two different strains) occurred in vinegars from cider and wine, whereas unique strains were found in spirit vinegars, which offered the most stressing conditions for bacterial growth.     

A RAPID,SIMPLE AND RELIABLE METHOD OF DIFFERENTIATING BREWING YEAST STRAINS BASED ON DNA RESTRICTION PATTERNS

DNA was isolated from polyploid brewing ale and lager yeast strains using a simple and rapid procedure which was a modification of a previously described method of Seehaus et al.¹⁴ The isolated DNA was cut with a number of restriction enzymes and subjected to agarose gel electrophoresis. Significant differences in banding patterns were observed between a Saccharomyces cerevisiae ale strain DNA and Saccharomyces uvarum (carlsbergensis) lager strain DNA, particularly with the enzyme Hpal. Differences were also observed between the banding patterns of digests from two ale strains, and from two lager strains. Use of this technique with appropriate restriction enzymes should prove useful for the rapid differentiation of brewing yeast strains.     

Antibiotic resistance,biochemical typing,and PFGE typing of <Emphasis Type="Italic">Bifidobacterium</Emphasis> strains commonly used in probiotic health foods

This study firstly analyzed the antibiotic resistance, biochemical typing, and pulsed-field gel electrophoresis typing of 45 Bifidobacterium strains commonly used in health foods. Most strains were resistant to antibiotics but their antibiotic resistance rates were not high: Fos (56.52%), TET (43.48%), CRO (21.74%), AMC (15.22%), GEN (13.04%), RIF (10.87%), CHL (8.7%), CTX (6.52%), VAN (4.35%), and ERY (4.35%). The 45 strains could be divided into 14 pulsed-field gel electrophoresis types, of which the strain numbers of six pulsed-field gel electrophoresis types were more than one. All the Bifidobacterium strains could be divided into nine types by API50CHL biochemical identification. The same species displayed same biochemical typings, expect for B. animalis. Furthermore, the results confirmed that the same pulsed-field gel electrophoresis-type strains had closer antibiotic resistance patterns, and the same biochemical-type strain also had similar antibiotic resistance patterns.     

High-resolution genotyping of Listeria monocytogenes by fluorescent amplified fragment length polymorphism analysis compared to pulsed-field gel electrophoresis, random amplified polymorphic DNA analysis, ribotyping, and PCR-restriction fragment length polymorphism analysis

The purpose of this study was to evaluate fluorescent amplified fragment length polymorphism (AFLP) analysis for the inter- and intraspecies differentiation of a collection of 96 strains of Listeria monocytogenes and 10 non-L. monocytogenes strains representing six other Listeria species of different origin. The AFLP technique was compared with three other molecular typing methods--ribotyping, random amplified polymorphic DNA analysis (RAPD), and pulsed-field gel electrophoresis (PFGE)--in terms of discriminatory ability. PCR-restriction fragment length polymorphism was included for virulence gene allele characterization. The 96 L. monocytogenes strains were divided into two major clusters by AFLP fingerprinting at a similarity level of 82% in concordance with the results of PFGE, RAPD, and ribotyping. One main cluster consisted of all of the 24 L. monocytogenes hly allele 1 strains, while another main cluster consisted of all of the 72 L. monocytogenes hly allele 2 strains. This indicates the existence of two distinct phylogenetic divisions. Isolates of the remaining Listeria species were not included in the clusters. AFLP, PFGE, and RAPD typing were highly discriminatory methods, with discrimination (D) indices of 0.974, 0.969, and 0.954, respectively, whereas ribotyping had a lower D index of 0.874. AFLP, PFGE, and RAPD typing showed some level of agreement in terms of strain grouping and differentiation. However, all three methods subdivided types of strains grouped by the other methods. Isolates with identical DNA profiles were distributed across the spectrum of origin. It was not possible to associate certain types with specific food sectors or clinical cases, which is indicative of the spread of L. monocytogenes clones across species. Overall, AFLP fingerprinting was suitable for the high-resolution genotyping of L. monocytogenes and had an equally high or higher differentiation power compared to PFGE or RAPD typing.     

一起纽波特沙门菌引起的食物中毒检测及菌株同源性分析

目的运用脉冲场凝胶电泳(PFGE)对一起食物中毒事件中分离的菌株进行同源性分析,为查明事件原因提供依据。方法采集患者、食品加工人员和剩余食物样本进行病原分离及鉴定,对分离到的菌株进行PFGE及耐药性分析。结果从采集的13份样本中检出4株纽波特沙门菌,其中1株来自病人样本,1株来自食品加工人员(厨师)样本,剩余牛肉、鸭肉各1株。4株菌株经PFGE分析,同源性为100%。4株分离菌株耐药谱相同。结论此次食物中毒由纽波特沙门菌引起,PFGE分型揭示菌株之间的流行病学联系,为事件的分析和溯源提供分子流行病学证据。     

Genetic characterization of commercial Saccharomyces cerevisiae isolates recovered from vineyard environments

One hundred isolates of the commercial Saccharomyces cerevisiae strain Zymaflore VL1 were recovered from spontaneous fermentations carried out with grapes collected from vineyards located close to wineries in the Vinho Verde wine region of Portugal. Isolates were differentiated based on their mitochondrial DNA restriction patterns and the evaluation of genetic polymorphisms was carried out by microsatellite analysis, interdelta sequence typing and pulsed-field gel electrophoresis (PFGE). Genetic patterns were compared to those obtained for 30 isolates of the original commercialized Zymaflore VL1 strain. Among the 100 recovered isolates we found a high percentage of chromosomal size variations, most evident for the smaller chromosomes III and VI. Complete loss of heterozygosity was observed for two isolates that had also lost chromosomal heteromorphism; their growth and fermentative capacity in a synthetic must medium was also affected. A considerably higher number of variant patterns for interdelta sequence amplifications was obtained for grape-derived strains compared to the original VL1 isolates. Our data show that the long-term presence of strain VL1 in natural grapevine environments induced genetic changes that can be detected using different fingerprinting methods. The observed genetic changes may reflect adaptive mechanisms to changed environmental conditions that yeast cells encounter during their existence in nature.